CHROMOMETHYLTRANSFERASE3/KRYPTONITE maintains the sulfurea paramutation in Solanum lycopersicum

Significance Paramutation involves the transfer of a repressive epigenetic mark between silent and active alleles. It is best known from exceptional non-Mendelian inheritance of conspicuous phenotypes in maize but also in other plants and animals. Recent genomic studies, however, indicate that paramutation may be less exceptional. It may be a consequence of wide-cross hybridization and may contribute to quantitative trait variation or unstable phenotypes in crops. Using the sulfurea (sulf) locus in tomato, we demonstrate that a self-reinforcing feedback loop involving DNA- and histone-methyl transferases CHROMOMETHYLTRANSFERASE3 (CMT3) and KRYPTONITE (KYP) is required for paramutation of sulf and that there is a change in chromatin organization. These findings advance the understanding of non-Mendelian inheritance in plants.

primers. Quantitative PCR was performed in reactions containing 1X Luna® Universal qPCR Master Mix (New England Biolabs) on a CFX384 system (Bio-Rad). qPCR was carried out denaturation step at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing/extension at 60°C for 30 sec. Relative expression was calculated using the ΔΔct method (2−ΔΔct) using the geometric mean of two reference genes (Table S3). Oligonucleotides used for expression analyses are listed in Table S3. sRNA-seq. Total RNA isolation was performed with the Direct-zol RNA Miniprep (Zymo Research) and Trizol reagent (Invitrogen) according to the manufacturer's instructions in leaf tissue of 4-week-old plants. For M82/sulf and F2 CMT3 sets, sRNA libraries were prepared using the NEBNext multiplex small RNA library prep kit (New England Biolabs) according to the manufacturer's instructions. Libraries were indexed during the PCR step with 12 cycles and size-selected using BluePippin. Pooled libraries were sequenced on a NextSeq500 (Illumina). NRPE1 library preparation and sequencing were outsourced to Novogene. Raw data (already demultiplexed and available in fastq format) was processed using the Snakemake pipeline available at https://github.com/sebmueller/snakemake_sRNAseq. Briefly, raw data was quality controlled using FastQC (v0.11.7) followed by 3' adaptor removal (trimming) using cutadapt removing Illumina universal adapters. All sequences <15 nt and >40 nt in length were discarded, and the remaining sequences mapped to the reference genome (Heinz 1706 genome version 3.0). Mapping was performed using Bowtie version 1.2 with uniquely mapping with "bowtie --wrapper basic-0 -v 0 -k 1 -m 1 --best -q" which only reports sRNAs mapping to unique locations. 0 mismatches were employed using bowtie version 1.2. DMR1 sRNA quantity was normalized as count per million (CPM) basing it on the total number of reads mapped at DMR1 (please see coordinates in Table S1) using deeptools version 3.3.1. The config.yaml file used for this analysis is supplied as Supplemental file S2.
Whole genome bisulphite sequencing. Genomic DNA was isolated from 100 mg leaf tissue 4-weekold plants using Dneasy Plant Mini Kit (Qiagen). Library preparation and sequencing were carried out by Novogene. In brief, DNA samples were fragmented into 200-400bp using Covaris S220. Terminal repairing, A-ligation, methylation sequencing adapters ligation were performed to the DNA fragments. Bisulphite treatment was carried out with Accel-NGS Methyl-Seq DNA Library Kit (IIllumina Cat No. 30096) followed by size selection and PCR amplification steps. Whole genome bisulphite processing raw data (paired-end; already demultiplexed and available in fastq format) was processed using the bisulfite Snakemake pipeline available at https://github.com/seb-mueller/snakemake-bisulfite. Briefly, raw data was quality controlled using FastQC (v0.11.7) followed by 3' adaptor removal (trimming) using trim_galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) removing Illumina universal adapters. Mapping and cytosine methylation calling was done using Bismark (3) on the reference genome (Heinz 1706 genome version 3.00) in CpG, CHG and CHH contexts. The config.yaml file used for this analysis is supplied as Supplemental file S3.

ChIP-qPCR.
Chromatin extraction was carried out as described in (1) using 1g of leaf tissue of 4 weekold plants. Chromatin was fragmented between 200-600bp in a Covaris E220 evolution (duty cycle: 20%, peak intensity: 140, cycles of burst: 200, time: 3 min). Immunoprecipitation was carried out as described in (1) with Anti-Histone H3 (di methyl K9) antibody -ChIP Grade (abcam, ab1220) and Anti-Histone H3 (tri methyl K4) antibody -ChIP Grade (abcam, ab8580). The material was reverse crosslinked by adding NaCl to a final concentration of 200mM and incubating at 65°C O/N. This was followed by a 30 min treatment with 1μL RNase A (Thermo Scientific) at 37°C and a 90 min treatment with 1.5 µl Proteinase K (Thermo Scientific) at 65°C. DNA was purified using the MinElute Kit (Qiagen) according to manufacturer's instructions and eluted in 35µl EB buffer. The eluted DNA was used to quantify enriched DNA fragments by standard qPCR methods using 1X Luna® Universal qPCR Master Mix (New England Biolabs) on a CFX384 system (Bio-Rad). Enrichment of DNA fragments for H3K9me2 and H3K4me3 analysis were calculated as % input (2(Ct input adjusted -Ct IP)*100) and normalised using the enrichment found for an unrelated reference locus CAC3. Oligonucleotides employed in this analysis are listed in Table S3.
Maize CHG subcontext analysis. For bisulphite sequencing, genomic maize DNA was extracted (6) from pools of 30-100 embryos or half of an ear. Bisulphite treatment was performed using 400ng of DNA and the EZ DNA Methylation-Gold kit (Zymo Research, D5006). The DNA regions of interest were PCR amplified (10 min 95°C, followed by 40 PCR cycles (30 sec 95°C, 30 sec appropriate annealing temp, 30 sec 72°C), and 5 min at 72°C). The PCR was performed using MethylTaq DNA polymerase (Diagenode, C09010010). For primer sequences, amplicon sizes and annealing temperatures see Table S5. In order to monitor for complete bisulphite conversion, a conversion control (Fie2 fragment) was amplified and analysed in each experiment (Fig S13) (similar to the -302 to -91 fragment described in (7)). PCR fragments were ligated into the pJET 1.2 vector (CloneJet PCR Cloning Kit, Thermo Scientific) according manufacturer's instructions. Positive Clones were identified by colony-PCR, Plasmid DNA was isolated from positive clones (GeneJet Plasmid Miniprep Kit, Thermo scientific) and subjected to Sanger sequencing. For the conversion control 14-16 clones were analysed and for each other fragment 22-31 clones were analysed. Frequency of DNA methylation at individual CHG motifs was inferred using Kismeth (8).
Hi-C. 0.5g leaf tissue of 4-week-old plants (per replicate) was fixed in 1% formaldehyde (v/v) and 0.01% Triton-X (v/v) for 20 min using vacuum infiltration. The reaction was quenched by adding glycine to a final concentration of 125 mM for 10 min using vacuum infiltration. The leaf tissue was rinsed 3 times with MilliQ water, pat dried and flash frozen in liquid Nitrogen. The in situ Hi-C library preparation was performed essentially as described in (9). For individual samples in each replicate was homogenized for preparing libraries. The libraries were sequenced on an Illumina NextSeq500 instrument with 2 x 75 bp reads. The Hi-C raw reads were mapped to Solanum lycopersicum genome Heinz 1706 assembly SL3.00 with an iterative mapping pipeline (9). The removal of PCR duplicates and reads filtering were performed as described in (9). Hi-C reads of each sample are summarised in Table S6. Hi-C map normalization with bin size 100kb was done using the "HiTC" package in R (10).

Data visualisation and statistical analysis
Plots for ChIP-seq, sRNA-seq and subcontext analysis were carried out using ggplot2 (11) and computationally reproducible scripts are available at https://github.com/claudiamartinho/Martinhoetal2021. Plots depicting DNA methylation levels determined by McRBC and expression levels were generated by employing the webtool PlotsOfData (12). Statistical tests were carried out in R using the function wilcox.test and p.adjust.method = "BH". Genome browser images were generated in IGV v2.7.2 (13).